Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis

Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.     

Phosphorylation of dedicator of cytokinesis 1 (Dock180) at tyrosine residue Y722 by Src family kinases mediates EGFRvIII-driven glioblastoma tumorigenesis

Glioblastoma, the most common primary malignant cancer of the brain, is characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. These traits cause glioblastomas to be highly resistant to current therapies with a resultant poor prognosis. Although aberrant oncogenic signaling driven by signature genetic alterations, such as EGF receptor (EGFR) gene amplification and mutation, plays a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here, we report that EGFRvIII (also known as ΔEGFR and de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in human glioblastoma, promotes tumorigenesis through Src family kinase (SFK)-dependent phosphorylation of Dock180, a guanine nucleotide exchange factor for Rac1. EGFRvIII induces phosphorylation of Dock180 at tyrosine residue 722 (Dock180(Y722)) and stimulates Rac1-signaling, glioblastoma cell survival and migration. Consistent with this being causal, siRNA knockdown of Dock180 or expression of a Dock180(Y722F) mutant inhibits each of these EGFRvIII-stimulated activities. The SFKs, Src, Fyn, and Lyn, induce phosphorylation of Dock180(Y722) and inhibition of these SFKs by pharmacological inhibitors or shRNA depletion markedly attenuates EGFRvIII-induced phosphorylation of Dock180(Y722), Rac1 activity, and glioblastoma cell migration. Finally, phosphorylated Dock180(Y722) is coexpressed with EGFRvIII and phosphorylated Src(Y418) in clinical specimens, and such coexpression correlates with an extremely poor survival in glioblastoma patients. These results suggest that targeting the SFK-p-Dock180(Y722)-Rac1 signaling pathway may offer a novel therapeutic strategy for glioblastomas with EGFRvIII overexpression.     

ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines

Therapies that target the EGF receptor (EGFR), such as gefitinib (IRESSA), are effective in a subset of patients with advanced non-small cell lung cancer (NSCLC). The differences in intracellular signaling networks between gefitinib-sensitive and -resistant NSCLCs remain poorly understood. In this study, we observe that gefitinib reduces phospho-Akt levels only in NSCLC cell lines in which it inhibits growth. To elucidate the mechanism underlying this observation, we compared immunoprecipitates of phosphoinositide 3-kinase (PI3K) between gefitinib-sensitive and -resistant NSCLC cell lines. We observe that PI3K associates with ErbB-3 exclusively in gefitinib-sensitive NSCLC cell lines. Gefitinib dissociates this complex, thereby linking EGFR inhibition to decreased Akt activity. In contrast, gefitinib-resistant cells do not use ErbB-3 to activate the PI3K/Akt pathway. In fact, abundant ErbB-3 expression is detected only in gefitinib-sensitive NSCLC cell lines. Two gefitinib-sensitive NSCLC cell lines with endogenous distinct activating EGFR mutations (L858R and Del747-749), frequently observed in NSCLC patients who respond to gefitinib, also use ErbB-3 to couple to PI3K. Down-regulation of ErbB-3 by means of short hairpin RNA leads to decreased phospho-Akt levels in the gefitinib-sensitive NSCLC cell lines, Calu-3 (WT EGFR) and H3255 (L858R EGFR), but has no effect on Akt activation in the gefitinib-resistant cell lines, A549 and H522. We conclude that ErbB-3 is used to couple EGFR to the PI3K/Akt pathway in gefitinib-sensitive NSCLC cell lines harboring WT and mutant EGFRs.     

Induction of tyrosine phosphorylation of Vav and expression of Pim-1 correlates with Jak2-mediated growth signaling from the erythropoietin receptor

The receptor for erythropoietin (Epo) belongs to the cytokine receptor family and lacks a tyrosine kinase domain. However, it has been hypothesized that a tyrosine kinase, Jak2, associates with the membrane proximal cytoplasmic region of Epo receptor (EpoR) and mediates the growth signaling from the receptor through tyrosine phosphorylation of cellular substrates. To explore the growth signaling pathways from the EpoR, we analyzed substrates of tyrosine phosphorylation induced by Epo stimulation in cells expressing various mutant EpoRs. The vav proto- oncogene product was found to be tyrosine phosphorylated after Epo stimulation in cells expressing the wild-type EpoR or a truncated receptor, H mutant, that retains the growth signaling function. In these cells, Epo also induced the expression of a serine/threonine kinase, Pim-1. However, Epo stimulation did not have any effect on Vav or Pim-1 in cells expressing a mutant EpoR, PM4 mutant, inactivated by a point mutation, Trp282 to Arg, in the membrane proximal region, which abrogates the interaction with Jak2. On the other hand, both tyrosine phosphorylation of Vav and expression of Pim-1 were observed constitutively in cells expressing a mutant EpoR that is constitutively activated by a point mutation, Arg 129 to Cys, in the extracellular domain. Jak2 was also constitutively tyrosine phosphorylated and activated in cells expressing this mutant, which confirms the crucial role of Jak2 in growth signaling from the EpoR. Taken together, these observations suggest that the tyrosine phosphorylation of Vav and the expression of Pim-1 may play important roles in growth signaling from the EpoR.     

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1beta (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.     

The carboxy-terminal region of the granulocyte colony-stimulating factor receptor transduces a phagocytic signal

Granulocyte colony-stimulating factor (G-CSF) induces proliferation, maturation, and functional activities of myeloid progenitors and mature neutrophils through a specific receptor, the G-CSF-R. Different signals are mediated by distinct regions of the cytoplasmic domain of G-CSF-R, but the precise role of each region has not yet been fully clarified. We evaluated the involvement of Syk kinase, essential in mediating phagocytic signals by Fcgamma receptors, in G-CSF-induced phagocytosis, using murine myeloid 32D cells transfected with wild-type (WT) human G-CSF-R (hG-CSF-R) or with a G-CSF-R mutant truncated at cytoplasmic amino acid 715. The G-CSF-R mutant lacks the immunoreceptor tyrosine-based activation motif (ITAM), putative binding site for Syk. Following treatment of WT hG-CSF-R transfectants with IgG-coated particles, there was a significant increase in phagocytosis in G-CSF-stimulated cells, in which Syk tyrosine phosphorylation occurred, paralleled by enhancement of its tyrosine kinase activity. In the mutant transfectants, no significant increase in phagocytosis or Syk tyrosine phosphorylation occurred after stimulation with G-CSF. We also demonstrated that tyrosine phosphorylation of the Src kinases Hck and Lyn occurs following G-CSF stimulation of cells expressing WT G-CSF-R, but that Hck is not phosphorylated in mutant G-CSF-R transfectants. The increase in phagocytosis following G-CSF stimulation cannot be attributed to a rapid de novo increase in expression of Fcgamma receptors. G-CSF induced expression of Fcgamma receptors only after prolonged stimulation. Our data provide evidence that the carboxy-terminal region of G-CSF-R plays a role in the phagocytosis of IgG-coated particles and that Syk and Hck kinase tyrosine phosphorylation is involved.     

Molecular imaging of active mutant L858R EGF receptor (EGFR) kinase-expressing nonsmall cell lung carcinomas using PET/CT

The importance of the EGF receptor (EGFR) signaling pathway in the development and progression of nonsmall cell lung carcinomas (NSCLC) is widely recognized. Gene sequencing studies revealed that a majority of tumors responding to EGFR kinase inhibitors harbor activating mutations in the EGFR kinase domain. This underscores the need for novel biomarkers and diagnostic imaging approaches to identify patients who may benefit from particular therapeutic agents and approaches with improved efficacy and safety profiles. To this goal, we developed 4-[(3-iodophenyl)amino]-7-{2-[2-{2-(2-[2-{2-([(18)F]fluoroethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}-ethoxy]-quinazoline-6-yl-acrylamide ([(18)F]F-PEG6-IPQA), a radiotracer with increased selectivity and irreversible binding to the active mutant L858R EGFR kinase. We show that PET with [(18)F]F-PEG6-IPQA in tumor-bearing mice discriminates H3255 NSCLC xenografts expressing L858R mutant EGFR from H441 and PC14 xenografts expressing EGFR or H1975 xenografts with L858R/T790M dual mutation in EGFR kinase domain, which confers resistance to EGFR inhibitors (i.e., gefitinib). The T790M mutation precludes the [(18)F]F-PEG6-IPQA from irreversible binding to EGFR. These results suggest that PET with [(18)F]F-PEG6-IPQA could be used for the selection of NSCLC patients for individualized therapy with small molecular inhibitors of EGFR kinase that are currently used in the clinic and have a similar structure (i.e., iressa, gefitinib, and erlotinib).     

Differential activation of epidermal growth factor (EGF) receptor downstream signaling pathways by betacellulin and EGF

To determine the downstream signaling pathways regulated by betacellulin (BTC) in comparison with epidermal growth factor (EGF), we used Chinese hamster ovary cells overexpressing the human EGF receptor (ErbB1/EGFR). The overall time-dependent activation of EGFR autophosphorylation was identical in cells treated with 1 nm BTC or 1.5 nm EGF. Analysis of site-specific EGFR phosphorylation demonstrated that the BTC and EGF tyrosine phosphorylation of Y1086 was not significantly different. In contrast, the autophosphorylation of Y1173 was markedly reduced in BTC-stimulated cells, compared with EGF stimulation that directly correlated with a reduced BTC stimulation of Shc tyrosine phosphorylation, Ras, and Raf-1 activation. On the other hand, Y1068 phosphorylation was significantly increased after BTC stimulation, compared with EGF in parallel with a greater extent of Erk phosphorylation. Expression of a dominant interfering MEK kinase 1 (MEKK1) and Y1068F EGFR more efficiently blocked the enhanced Erk activation by BTC, compared with EGF. Interestingly BTC had a greater inhibitory effect on apoptosis, compared with EGF, and expression of Y1068F EGFR abolished this enhanced inhibitory effect. Together, these data indicated that although BTC and EGF share overlapping signaling properties, the ability of BTC to enhance Erk activation occurs independent of Ras. The increased BTC activation results from a greater extent of Y1068 EGFR tyrosine phosphorylation and subsequent increased recruitment of the Grb2-MEKK1 complex to the plasma membrane, compared with EGF stimulation. The increased Erk activation by BTC associated with antiapoptotic function.     

In vitro tyrosine phosphorylation studies on RAS proteins and calmodulin suggest that polylysine-like basic peptides or domains may be involved in interactions between insulin receptor kinase and its substrate.

We have investigated the in vitro tyrosine phosphorylation of the HRAS and KRAS proteins by human placental insulin receptor kinase. Purified HRAS proteins are not phosphorylated by purified insulin receptor kinase. Since the tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro requires cofactors such as protamine and poly(L-lysine), we examined the possibility that poly(L-lysine) may also potentiate the interaction between RAS proteins and the insulin receptor. We found that purified HRAS proteins are indeed phosphorylated by purified insulin receptor kinase in the presence of poly(L-lysine). In contrast, the KRAS protein, which carries an extremely basic domain (residues 172-182, Lys-Asp-Glu-Lys6-Ser-Arg), is phosphorylated by the receptor kinase without the addition of basic proteins. We then determined whether the KRAS basic domain peptide plays a role similar to that of poly(L-lysine) and found that both the HRAS protein and calmodulin are phosphorylated by the receptor kinase in the presence of the KRAS basic domain peptide. Further examination of the role of poly(L-lysine) in potentiating tyrosine phosphorylation of the HRAS protein and calmodulin by purified insulin receptor kinase indicates that poly(L-lysine) affects the conformation of these protein substrates as well as that of the receptor kinase domain. These studies suggest that polylysine-like basic proteins or domains are required to establish the interaction between insulin receptor kinase and its substrate.     

Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.     

Diverse injurious stimuli reduce protein tyrosine phosphatase-μ expression and enhance epidermal growth factor receptor signaling in human airway epithelia

In response to injury, airway epithelia utilize an epidermal growth factor (EGF) receptor (EGFR) signaling program to institute repair and restitution. Protein tyrosine phosphatases (PTPs) counterregulate EGFR autophosphorylation and downstream signaling. PTPμ is highly expressed in lung epithelia and can be localized to intercellular junctions where its ectodomain homophilically interacts with PTPμ ectodomain expressed on neighboring cells. We asked whether PTPμ expression might be altered in response to epithelial injury and whether altered PTPμ expression might influence EGFR signaling. In A549 cells, diverse injurious stimuli dramatically reduced PTPμ protein expression. Under basal conditions, small interfering RNA (siRNA)-induced silencing of PTPμ increased EGFR Y992 and Y1068 phosphorylation. In the presence of EGF, PTPμ knockdown increased EGFR Y845, Y992, Y1045, Y1068, Y1086, and Y1173 but not Y1148 phosphorylation. Reduced PTPμ expression increased EGF-stimulated phosphorylation of Y992, a docking site for phospholipase C (PLC)γ(1), activation of PLCγ(1) itself, and increased cell migration in both wounding and chemotaxis assays. In contrast, overexpression of PTPμ decreased EGF-stimulated EGFR Y992 and Y1068 phosphorylation. Therefore, airway epithelial injury profoundly reduces PTPμ expression, and PTPμ depletion selectively increases phosphorylation of specific EGFR tyrosine residues, PLCγ(1) activation, and cell migration, providing a novel mechanism through which epithelial integrity may be restored.     

G-CSF-induced tyrosine phosphorylation of Gab2 is Lyn kinase dependent and associated with enhanced Akt and differentiative, not proliferative, responses

The granulocyte colony-stimulating factor receptor (G-CSFR) transduces intracellular signals for myeloid cell proliferation, survival, and differentiation through the recruitment of nonreceptor protein tyrosine kinases Lyn and janus kinase 2 (Jak2). This results in the tyrosine phosphorylation of a small set of positive and negative adapters and effectors. Grb2-associated binder-2 (Gab2) is a newly described adapter molecule, preferentially expressed in hematopoietic cells and associated with phosphatidylinositol 3 (PI3) kinase. Studies suggest that Gab2 plays both positive and negative roles in cytokine receptor signaling. To investigate the role Gab2 plays in G-CSF receptor-mediated signaling, we have analyzed its activation state and correlated that with wild-type and mutant G-CSF receptors stably expressed in the murine factor-dependent Ba/F3 cell lines. G-CSF-induced tyrosine phosphorylation of Gab2 occurred in the wild-type and single Y-to-F mutants (Y704F, Y729F, and Y744F), but not in the ADA and W650R loss-of-function mutants. Cells expressing truncated proximal G-CSFR, the tyrosine-null (Y4F) G-CSFR, or Y764F mutant receptors had decreased phosphorylation of Gab2. Specific inhibitors of Src kinase (PD173 and PP1) but not Jak2 kinase (AG490) blocked Gab2 phosphorylation. Phosphorylation of Gab2 occurred in wild-type, but not Lyn-deficient, G-CSFR-transfected DT40 B cells. These data propose that Lyn, not Jak2, phosphorylates Gab2 and that maximal phosphorylation of Gab2 requires Y764, a Grb2-binding site. Serine phosphorylation of Akt, a marker of PI3-kinase activity, was detected in both wild-type and truncated proximal domain receptors, but not in the ADA and W650R mutants. Levels of phospho-Akt and phospho-extracellular signal-regulated kinase (phospho-ERK) were greater in proximal truncated than in wild-type G-CSFR cells, suggesting that Gab2 is dissociated from PI3 kinase or ERK activities. Overexpression of Gab2 enhanced the phosphorylation state of Akt, but not of ERK. This inhibited the proliferation of wild-type and truncated G-CSFR-transfected Ba/F3 cells and enhanced their myeloid differentiation. All together, these data indicate that G-CSF treatment leads to Lyn-mediated tyrosine phosphorylation of Gab2, which may serve as an important intermediate of enhanced Akt activity and myeloid differentiation, not growth/survival response.     

Phosphoproteomic analysis of Her2/neu signaling and inhibition

Her2/neu (Her2) is a tyrosine kinase belonging to the EGF receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. We sought to characterize Her2 signal transduction pathways further by using MS-based quantitative proteomics. Stably transfected cell lines overexpressing Her2 or empty vector were generated, and the effect of an EGFR and Her2 selective tyrosine kinase inhibitor, PD168393, on these cells was characterized. Quantitative measurements were obtained on 462 proteins by using the SILAC (stable isotope labeling with amino acids in cell culture) method to monitor three conditions simultaneously. Of these proteins, 198 showed a significant increase in tyrosine phosphorylation in Her2-overexpressing cells, and 81 showed a significant decrease in phosphorylation. Treatment of Her2-overexpressing cells with PD168393 showed rapid reversibility of the majority of the Her2-triggered phosphorylation events. Phosphoproteins that were identified included many known Her2 signaling molecules as well as known EGFR signaling proteins that had not been previously linked to Her2, such as Stat1, Dok1, and delta-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site in Her2, Y877, which is located in the activation loop of the kinase domain, is distinct from the known C-terminal tail autophosphorylation sites, and may have important implications for regulation of Her2 signaling. Network modeling, which combined phosphoproteomic results with literature-curated protein-protein interaction data, was used to suggest roles for some of the previously unidentified Her2 signaling proteins.     

Signaling networks assembled by oncogenic EGFR and c-Met

A major question regarding the sensitivity of solid tumors to targeted kinase inhibitors is why some tumors respond and others do not. The observation that many tumors express EGF receptor (EGFR), yet only a small subset with EGFR-activating mutations respond clinically to EGFR inhibitors (EGFRIs), suggests that responsive tumors uniquely depend on EGFR signaling for their survival. The nature of this dependence is not understood. Here, we investigate dependence on EGFR signaling by comparing non-small-cell lung cancer cell lines driven by EGFR-activating mutations and genomic amplifications using a global proteomic analysis of phospho-tyrosine signaling. We identify an extensive receptor tyrosine kinase signaling network established in cells expressing mutated and activated EGFR or expressing amplified c-Met. We show that in drug sensitive cells the targeted tyrosine kinase drives other RTKs and an extensive network of downstream signaling that collapse with drug treatment. Comparison of the signaling networks in EGFR and c-Met-dependent cells identify a "core network" of approximately 50 proteins that participate in pathways mediating drug response.     

Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation.Epidermal growth factor receptor (EGFR), one of the most studied receptor tyrosine kinases, is a drug target for cancer therapy, because its kinase activity correlates with tumorigenicity (1). Under normal conditions, EGFR forms dimers upon ligand binding and induces kinase activation (2–6). The conformational change of EGFR from tethered to extended form induced by ligand binding involves the exposure of the interface, followed by dimerization, activation, and autophosphorylation (7). The phosphorylation code of EGFR determines the propensity of the downstream signaling network to regulate cell proliferation, survival, migration, and angiogenesis (8, 9).In a significant fraction of patients with nonsmall cell lung cancer (NSCLC), especially patients in Asia and those with the adenocarcinoma subtype, mutations in the kinase domain of EGFR cause constitutive activation and have been identified as an important factor in EGFR dysregulation (10, 11). Particularly, mutation from leucine to arginine at position 858 (L858R) and, less significantly, deletion of exon 19 that eliminates four amino acids (LREA) account for ∼90% of the mutations involved in the constitutive activation of EGFR. These mutations are commonly found in patients with increased sensitivity to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib (12–14). However, most patients with such mutations show resistance within months after TKI therapy, and >50% of them develop a second EGFR mutation, T790M, which confers TKI resistance by increasing the affinity for ATP and decreasing the affinity for TKIs (15–17).Studies have demonstrated that the glycans on EGFR participate in the regulation of EGFR function. The number of N-glycans and the degree of branching can regulate the cell-surface expression of EGFR in response to N-acetyl-d-glucosamine (GlcNAc) supplementation (18). In addition, studies with site-directed mutagenesis indicate that the glycans on Asn420 and 579 prevent EGFR from ligand-independent dimerization (19–21), and knocking down/out fucosyltransferase 8, the enzyme responsible for the core fucosylation, attenuates EGFR phosphorylation and EGF binding (22, 23). Moreover, our previous study revealed that sialylation and fucosylation suppress EGFR dimerization, autophosphorylation, and EGF-induced lung cancer cell invasion (24).Here, we investigated the effect of sialylation on EGFR dimerization to understand how extracellular sialylation influences intracellular phosphorylation in both wild-type and mutant EGFR. Our biochemical data demonstrated that sialylation could suppress EGFR dimerization by attenuating its association with EGF, and sialylation could significantly and selectively suppress tyrosine phosphorylation and affect the levels of phosphoserine and phosphothreonine on EGFR. In EGFR mutants, especially L858R/T790M, sialylation was observed to have a selective effect on EGFR phosphorylation, and inhibition of sialylation resulted in increased phosphorylation and resistance to gefitinib in this TKI-resistant lung cancer cell line. Further study of these findings should provide a better understanding of EGFR-mediated phosphorylation and disease progression affected by glycosylation and lead to the development of a new therapeutic strategy.     

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.     

Dihydrotestosterone upregulates the expression of epidermal growth factor receptor and ERBB2 in androgen receptor-positive bladder cancer cells

Androgen receptor (AR) signals play important roles in bladder carcinogenesis and tumor progression. Activation of the epidermal growth factor receptor (EGFR) family, including EGFR and ERBB2, leads to bladder cancer cell growth and correlates with poor patients' prognosis. However, cross talk between AR and EGFR/ERBB2 pathways in bladder cancer remains poorly understood. In AR-positive bladder cancer UMUC3 and TCC-SUP cells, dihydrotestosterone (DHT) increased the expression of EGFR and ERBB2 both in mRNA and in protein levels, and an anti-androgen hydroxyflutamide antagonized the effect of DHT. The necessity of AR was confirmed by silencing the receptor, using short hairpin RNA (shRNA), in UMUC3 cells, as well as by expressing the receptor in AR-negative 5637 cells. Of note were much higher basal levels of EGFR and ERBB2 in UMUC3-control-shRNA than in UMUC3-AR-shRNA and those of EGFR in 5637-AR than in 5637-V. DHT additionally upregulated the levels of phosphorylation of EGFR (pEGFR) and its downstream proteins AKT (pAKT) and ERK1/2 (pERK), induced by EGF treatment, in AR-positive cells. Immunohistochemistry on cystectomy specimens showed strong associations between expressions of AR and EGFR (P=0.0136), pEGFR (P=0.0041), ERBB2 (P=0.0331), or pERK (P=0.0274), but not of pAKT (P=0.5555). The Kaplan-Meier and log-rank tests further revealed that positivity of AR (P=0.0005), EGFR (P=0.2425), pEGFR (P=0.1579), ERBB2 (P=0.2997), or pERK (P=0.1270) and negativity of pAKT (P=0.0483) were associated with tumor progression. Our results indicate that AR activation upregulates the expression of EGFR and ERBB2 in bladder cancer cells. AR signals may thus contribute to the progression of bladder cancer via regulation of the EGFR/ERBB2 pathways.     

Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478

Blockade of epidermal growth factor receptor (EGFR) signaling with specific inhibitors of the EGFR tyrosine kinase retards cellular proliferation and arrests the growth of tumor xenografts. AG1478, an inhibitor of the EGFR tyrosine kinase, is used in laboratory studies; however, its therapeutic potential has not been elucidated. Therefore, we evaluated an aqueous form of AG1478 for its antitumor activity in mice bearing human xenografts expressing the WT EGFR or a naturally occurring ligand-independent truncation of the EGFR [delta2-7 (de2-7) EGFR or EGFRvIII]. Parenteral administration of soluble AG1478 blocked phosphorylation of the EGFR at the tumor site and inhibited the growth of A431 xenografts that overexpress the WT EGFR and glioma xenografts expressing the de2-7 EGFR. Strikingly, even subtherapeutic doses of AG1478 significantly enhanced the efficacy of cytotoxic drugs, with the combination of AG1478 and temozolomide displaying synergistic antitumor activity against human glioma xenografts. AG1478 was also examined in combination with mAb 806, an anti-EGFR antibody that was raised against the de2-7 EGFR but unexpectedly also binds a subset of the EGFR expressed in cells exhibiting amplification of the EGFR gene. The combination of AG1478 and mAb 806 displayed additive, and in some cases synergistic, antitumor activity against tumor xenografts overexpressing the EGFR. Here, we demonstrate that different classes of inhibitors to the EGFR can have synergistic antitumor activity in vivo. These results establish the antitumor efficacy of the EGFR inhibitor AG1478 and provide a rationale for its clinical evaluation in combination with both chemotherapy and other EGFR therapeutics.